Location-Specific Epigenetic Regulation of the Metallothionein 3 Gene in Esophageal Adenocarcinomas

Dunfa Peng,Mary Kay Washington,Aixiang Jiang,Wael El-Rifai,Christopher A Moskaluk,Regine Schneider-Stock,Tian-Ling Hu,Hassan Ashktorab

doi:10.1371/journal.pone.0022009

Dunfa Peng, Mary Kay Washington + Show 6 more

Open Access

https://doi.org/10.1371/journal.pone.0022009

Copy DOI

Abstract

BackgroundMetallothionein 3 (MT3) maintains intracellular metal homeostasis and protects against reactive oxygen species (ROS)-induced DNA damage. In this study, we investigated the epigenetic alterations and gene expression of the MT3 gene in esophageal adenocarcinomas (EACs).Methods and ResultsUsing quantitative bisulfite pyrosequencing, we detected unique DNA methylation profiles in the MT3 promoter region. The CpG nucleotides from −372 to −306 from the transcription start site (TSS) were highly methylated in tumor (n = 64) and normal samples (n = 51), whereas CpG nucleotides closest to the TSS (−4 and +3) remained unmethylated in all normal and most tumor samples. Conversely, CpG nucleotides in two regions (from −139 to −49 and +296 to +344) were significantly hypermethylated in EACs as compared to normal samples [FDR<0.001, −log10(FDR)>3.0]. The DNA methylation levels from −127 to −8 CpG sites showed the strongest correlation with MT3 gene expression (r = −0.4, P<0.0001). Moreover, the DNA hypermethylation from −127 to −8 CpG sites significantly correlated with advanced tumor stages and lymph node metastasis (P = 0.005 and P = 0.0313, respectively). The ChIP analysis demonstrated a more repressive histone modification (H3K9me2) and less active histone modifications (H3K4me2, H3K9ace) in OE33 cells than in FLO-1 cells; concordant with the presence of higher DNA methylation levels and silencing of MT3 expression in OE33 as compared to FLO-1 cells. Treatment of OE33 cells with 5-Aza-deoxycitidine restored MT3 expression with demethylation of its promoter region and reversal of the histone modifications towards active histone marks.ConclusionIn summary, EACs are characterized by frequent epigenetic silencing of MT3. The choice of specific regions in the CpG island is a critical step in determining the functional role and prognostic value of DNA methylation in cancer cells.

Highlights

Esophageal adenocarcinoma (EAC) is one of the human malignancies with the fastest growing incidence rates in the Western world [1,2,3]
In summary, EACs are characterized by frequent epigenetic silencing of Metallothionein 3 (MT3)
Our analysis indicated that the human MT3 gene contains a large CpG island located from 2372 bp upstream of the transcript start site (TSS) to approximately +344 bp downstream of the transcription start site (TSS) (Figure 1A)

Summary

Introduction

Esophageal adenocarcinoma (EAC) is one of the human malignancies with the fastest growing incidence rates in the Western world [1,2,3]. Chronic gastro-esophageal reflux disease (GERD), with accumulation of reactive oxygen species (ROS) and subsequent oxidative DNA damage, is one of the main risk factors for the development of BE and its progression to adenocarcinoma [7,8,9,10]. In addition to DNA methylation, other epigenetic alterations such as histone modifications, histone acetylation, and methylation are involved in the regulation of gene expression [13,14,15]. Epigenetic cross talk between DNA methylation and histone modifications occurs through various physiologic and pathologic conditions. The combination of these dynamic interactions determines gene expression [16,17]. We investigated the epigenetic alterations and gene expression of the MT3 gene in esophageal adenocarcinomas (EACs)

Methods

Results

Discussion

Conclusion